pH sensitive fluorescent dyes employed in biological research and medical diagnostics belong to two groups, each distinguished by the origin of fluorescent responses to changes in pH. The first group includes compounds having fluorescence controlled by the ionization of phenolic hydroxyl groups in a fluorophore. Examples include fluorescein, carboxyfluorescein, Oregon Green®, SNARF®, SNAFL®, and HPTS indicators.
U.S. Patent Publication No. 2006/0051874 (M. W. Reed, et al) describes fluorescein-like structures incorporated into a fluorescent detector for monitoring pH of the blood in bank storages. Because the degree of ionization of these type molecules increase upon lowering the acidity of the environment, they become more fluorescent as pH increases.
Fluorescent pH sensors of the second group include an amino group (aliphatic or aromatic) as an indicator moiety along with a reporter fluorescent dye moiety. When such a molecule adsorbs a photon, creating an excited electronic state, the electron of the amino group's unshared pair transfers to the orbital vacated by excitation. Such an electron transfer, referred to as Photoinduced Electron Transfer (PET) prevents the excited molecule from emission transition, thus the fluorescence of the dye is quenched. Protonation of the amino group changes the nature and energy of the pair's orbital and stops the PET. As a result, the fluorescent reporter moiety responds to a pH change. Because protonation of the amino group cancels the quenching, the PET-based sensors become more fluorescent as pH decreases.
Examples of PET-based pH sensors include LysoSensor dyes, which contain dimethylamino group as an indicator moiety and CypHer® 5E dye having an indolenine indicator group. One disadvantage of these sensors is that the working range is shifted to the acidic side because of the low pKa of the indicator amino group.
A family of rhodamine-based pH sensors are described in WO 2005/098437. The dyes have a benzene ring substituted ortho to the xanthene moiety by —OH or —SH (or their depronated forms) and WO 2005/098437 states that the —OH or —SH is believed responsible for the pH response of the dye. These dyes display a pH-dependency similar to amine PET indicators but were designed to have pKa values of less than 6 based on a perceived need for a pH sensor that would target cell compartments with a pH of less than 6. The WO 2005/098437 application purports that the strong electron withdrawing properties of the tetramethylrhodamine moiety in the dyes, significantly decreases the pKa of the indicator group, thus shifting sensors' working range toward highly acidic pH values. However, this thereby limits the applicability of the dyes described in WO 2005/098 at a physiological pH (e.g pH 6-7), especially in biological systems. These prior art compounds have been found by us to have potentially inconvenient instability in solution.
Accordingly, there is a need for additional pH sensitive fluorescent dyes, advantageously with improved properties, including in at least some compounds the ability to detect pH changes in biological systems. It is an object of the present invention to develop a novel class of fluorescent pH sensors, desirably mitigating or removing the disadvantages of the compounds known in art. Particularly, it is an object of aspects of the present invention to provide a relatively stable class of pH sensors, preferably with a working range towards neutral and other biologically relevant pH values.